LOCUS       sequence-61239-e        6433 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6433)
  AUTHORS   Gupta A, Hall VL, Kok FO, Shin M, McNulty JC, Lawson ND, Wolfe SA
  TITLE     Targeted Chromosomal Deletions and Inversions in Zebrafish.
  JOURNAL   Genome Res. 2013 Mar 11.
  PUBMED    23478401
REFERENCE   2  (bases 1 to 6433)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Genome Res.
            2013 Mar 11."
COMMENT     Sequence Label: pJDS71-GG-SHD
FEATURES             Location/Qualifiers
     source          1..6433
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             907..972
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             979..999
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1694..2281
                     /codon_start=1
                     /product="nonspecific DNA cleavage domain of the FokI 
                     endonuclease (Li et al., 1992)"
                     /label=FokI cleavage domain
                     /note="must dimerize to be catalytically active"
                     /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     CDS             2297..2338
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2348..2365
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2388..2405)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2394..2618
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2664..3092
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2751..2770)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2961..2982
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3101..3121)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3106..3436
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3287..3422
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3349..3368
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3484..3531
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3550..3948
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4106..4227
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4143..4162)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4197..4216
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4276..4292)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4276..4292)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4289..4311)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4300..4316
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4324..4330,4331..4348,4349..4354))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4369..4390
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4507..4524)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4678..5266)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4758..4777)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5437..6228,6229..6297))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6060..6079
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6298..6402)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 accatggact acaaagacca tgacggtgat tataaagatc atgacatcga ttacaaggat
      961 gacgatgaca agatggcccc caagaagaag aggaaggtgg gcattcaccg cggggtacct
     1021 atggtggact tgaggacact cggttattcg caacagcaac aggagaaaat caagcctaag
     1081 gtcaggagca ccgtcgcgca acaccacgag gcgcttgtgg ggcatggctt cactcatgcg
     1141 catattgtcg cgctttcaca gcaccctgcg gcgcttggga cggtggctgt caaataccaa
     1201 gatatgattg cggccctgcc cgaagccacg cacgaggcaa ttgtaggggt cggtaaacag
     1261 tggtcgggag cgcgagcact tgaggcgctg ctgactgtgg cgggtgagct tagggggcct
     1321 ccgctccagc tcgacaccgg gcagctgctg aagatcgcga agagaggggg agtaacagcg
     1381 gtagaggcag tgcacgcctg gcgcaatgcg ctcaccgggg cccccttgcc cctggagacg
     1441 attaatcacc ggtcgtctcc ctatcgcttc ccacgacgga ggacggccag ccttggagtc
     1501 catcgtagcc caattgtcca ggcccgatcc cgcgttggct gcgttaacga atgaccatct
     1561 ggtggcgttg gcatgtcttg gtggacgacc cgcgctcgat gcagtcaaaa agggtctgcc
     1621 tcatgctccc gcattgatca aaagaaccaa ccggcggatt cccgagagaa cttcccatcg
     1681 agtcgcggga tcccaactag tcaaaagtga actggaggag aagaaatctg aacttcgtca
     1741 taaattgaaa tatgtgcctc atgaatatat tgaattaatt gaaattgcca gaaattccac
     1801 tcaggataga attcttgaaa tgaaggtaat ggaatttttt atgaaagttt atggatatag
     1861 aggtaaacat ttgggtggat caaggaaacc ggacggagca atttatactg tcggatctcc
     1921 tattgattac ggtgtgatcg tggatactaa agcttatagc ggaggttata atctgccaat
     1981 tggccaagca gatgaaatgc aacgatatgt cgaagaaaat caaacacgaa acaaacatat
     2041 caaccctaat gaatggtgga aagtctatcc atcttctgta acggaattta agtttttatt
     2101 tgtgagtggt cactttaaag gaaactacaa agctcagctt acacgattaa atcatatcac
     2161 taattgtaat ggagctgttc ttagtgtaga agagctttta attggtggag aaatgattaa
     2221 agccggcaca ttaaccttag aggaagtcag acggaaattt aataacggcg agataaactt
     2281 ttaagggccc ttcgaaggta agcctatccc taaccctctc ctcggtctcg attctacgcg
     2341 taccggtcat catcaccatc accattgagt ttaaacccgc tgatcagcct cgactgtgcc
     2401 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     2461 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     2521 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     2581 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     2641 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2701 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2761 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2821 catcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2881 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2941 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     3001 ctcggtctat tcttttgatt tataagggat tttggggatt tcggcctatt ggttaaaaaa
     3061 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     3121 tgtggaaagt ccccaggctc cccaggcagg cagaagtatg caaagcatgc atctcaatta
     3181 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3241 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     3301 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     3361 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     3421 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca
     3481 cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg
     3541 aactaaacca tggccaagcc tttgtctcaa gaagaatcca ccctcattga aagagcaacg
     3601 gctacaatca acagcatccc catctctgaa gactacagcg tcgccagcgc agctctctct
     3661 agcgacggcc gcatcttcac tggtgtcaat gtatatcatt ttactggggg accttgtgca
     3721 gaactcgtgg tgctgggcac tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc
     3781 gcgatcggaa atgagaacag gggcatcttg agcccctgcg gacggtgtcg acaggtgctt
     3841 ctcgatctgc atcctgggat caaagcgata gtgaaggaca gtgatggaca gccgacggca
     3901 gttgggattc gtgaattgct gccctctggt tatgtgtggg agggctaagc acttcgtggc
     3961 cgaggagcag gactgacacg tgctacgaga tttcgattcc accgccgcct tctatgaaag
     4021 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     4081 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     4141 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4201 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     4261 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4321 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4381 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4441 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     4501 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4561 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4621 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4681 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4741 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4801 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4861 ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4921 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     4981 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5041 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5101 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5161 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5221 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5281 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5341 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5401 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5461 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5521 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5581 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5641 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5701 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5761 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5821 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5881 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     5941 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6001 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6061 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6121 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6181 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6241 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6301 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6361 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     6421 gccacctgac gtc
//
